Creation of photocyclic vertebrate rhodopsin by single amino acid substitution

  1. Kazumi Sakai
  2. Yoshinori Shichida
  3. Yasushi Imamoto
  4. Takahiro Yamashita  Is a corresponding author
  1. Department of Biophysics, Graduate School of Science, Kyoto University, Japan
  2. Research Organization for Science and technology, Ritsumeikan University, Japan

Abstract

Opsins are universal photoreceptive proteins in animals and can be classified into three types based on their photoreaction properties. Upon light irradiation, vertebrate rhodopsin forms a metastable active state, which cannot revert back to the original dark state via either photoreaction or thermal reaction. By contrast, after photoreception, most opsins form a stable active state which can photoconvert back to the dark state. Moreover, we recently found a novel type of opsins whose activity is regulated by photocycling. However, the molecular mechanism underlying this diversification of opsins remains unknown. In this study, we showed that vertebrate rhodopsin acquired the photocyclic and photoreversible properties upon introduction of a single mutation at position 188. This revealed that the residue at position 188 contributes to the diversification of photoreaction properties of opsins by its regulation of the recovery from the active state to the original dark state.

Editor's evaluation

This manuscript describes an investigation of the evolution of monostable rhodopsins, typically found in vertebrates. It highlights that single amino acid changes in vertebrate rhodopsins can create a partial bistable retinal pigment that can be photoconverted back to the ground state or it will slowly convert back to the ground state retinal isomer. The rationale for the experiments came from the discovery of a very interesting activation mechanism of the nonvisual pigment Opn5L1. This work has important implications for how our visual pigments have been optimized during evolution, and it contributes important insights into engineering bistable pigments for optogenetic applications.

https://doi.org/10.7554/eLife.75979.sa0

Introduction

Opsins are photosensitive G-protein-coupled receptors and are universally found in diploblastic and triploblastic animals. All opsins share common structural elements including seven transmembrane domains and bind a light-absorbing chromophore, retinal, via a Schiff base linkage to Lys296 (based on the bovine rhodopsin numbering system) of opsin. Opsins function for both visual and nonvisual photoreception and are classified into several groups based on their amino acid sequence (Shichida and Matsuyama, 2009; Koyanagi and Terakita, 2014). Bovine rhodopsin is the best-studied opsin (Yau and Hardie, 2009) and it functions as a visual photoreceptive protein in the retina and binds 11-cis retinal in the dark. Photoisomerization of retinal to the all-trans form produces the meta II intermediate of rhodopsin, which couples with G protein. Meta II is a metastable active state and spontaneously converts to meta III (Heck et al., 2003). In addition, light irradiation of meta II induces the formation of meta III rather than the original dark state (Bartl et al., 2001; Ritter et al., 2008). That is, the active state meta II very inefficiently converts back to the original dark state by photoreaction or thermal reaction. These observations show that vertebrate rhodopsin is specialized for photoactivation, and is thus characterized as a monostable opsin. By contrast, mollusk and arthropod rhodopsins form a stable active state, the acid-meta state, by photoisomerization of 11-cis to all-trans retinal, and the active state can photoconvert back to the original dark state, which contains 11-cis retinal (Koyanagi and Terakita, 2014; Yau and Hardie, 2009). That is, these opsins have two stable states, the dark and active states, which are interconvertible by light and thus are known as bistable opsins. Recent accumulation of knowledge about the molecular properties of opsins revealed that many members of various opsin groups are bistable opsins (Figure 1—figure supplement 1A), which suggests that vertebrate rhodopsin evolved as a monostable opsin from an ancestral bistable opsin (Shichida and Matsuyama, 2009).

Recently, we identified a novel type of opsin, Opn5L1, as a photocycle opsin (Sato et al., 2018). Opn5L1 binds all-trans retinal, not 11-cis retinal, to form the active state in the dark. Light irradiation suppresses the G protein activation ability of Opn5L1 by the photoisomerization of the retinal to 11-cis form. Subsequent formation of a covalent adduct between the retinal and Cys188 of the opsin induces the conversion of the C11 = C12 double bond to a single bond in the retinal. Afterward, the thermal rotation of the C11–C12 single bond in the retinal results in dissociation of the Cys188-retinal adduct and regeneration of the original dark state. The combination of photoisomerization and thermal isomerization of retinal regulates the ability of Opn5L1 to activate G protein, making this the first animal opsin whose activity is controlled by its photocyclic reaction.

Comparison of the amino acid sequences among opsins shows that the cysteine residue at position 188 is well conserved in the Opn5L1 group but rarely found in other opsin groups (Sato et al., 2018; Yamashita, 2020), which supports the importance of Cys188 for the unique photocyclic reaction of Opn5L1. On the other hand, vertebrate rhodopsin and cone pigments, which are characterized as monostable opsins, have a glycine residue at this position (Figure 1—figure supplement 1A). In this study, we analyzed whether the mutation at position 188 (Figure 1—figure supplement 1B) can make bovine rhodopsin photocyclic. Our detailed analysis revealed that G188C mutant photoconverted to the active state, meta II, which thermally recovered to the original dark state. In addition, light irradiation of meta II of G188C mutant induced reversion to the original dark state. Therefore, G188C mutant of bovine rhodopsin exhibits the photocyclic and photoreversible property and the residue at position 188 regulates the recovery from the active state to the original dark state in vertebrate rhodopsin.

Results and discussion

Acquisition of photocyclic property of bovine rhodopsin G188C mutant

In a previous report, we revealed that Opn5L1 possesses a cysteine residue at position 188, which underlies the photocyclic reaction of the opsin (Sato et al., 2018). Thus, to analyze whether G188C mutant of bovine rhodopsin acquires the photocyclic property, we purified G188C mutant after reconstitution with 11-cis retinal. However, we found that G188C mutant has much lower thermal stability than wild-type. That is, G188C mutant gradually decayed during incubation in the dark at 37°C (Figure 1B), whereas wild-type was quite stable under the same conditions (Figure 1A). Therefore, we improved the thermal stability of G188C mutant to analyze the detailed molecular properties of the mutant. Following previous reports (Xie et al., 2003; Standfuss et al., 2007), we introduced two cysteine residues (N2C/D282C) into the mutant and measured the thermal decay rate during incubation in the dark at 37°C. The time-dependent spectral changes showed that this G188C/N2C/D282C mutant decayed much more slowly than G188C mutant (Figure 1C). Therefore, we compared the spectral changes among wild-type, N2C/D282C and G188C/N2C/D282C mutant at 20°C. The spectrum of wild-type was shifted into the UV region after yellow light irradiation (Figure 1—figure supplement 2A), which is indicative of the formation of meta II intermediate containing all-trans-15-anti retinal (Figure 1D). Subsequently, the absorbance at around 470 nm increased, which indicated the transition from meta II to meta III intermediate containing all-trans-15-syn retinal (Vogel et al., 2003; Figure 1D). These spectral changes were also observed in N2C/D282C (Figure 1E) as noted in the previous report (Xie et al., 2003). By contrast, G188C/N2C/D282C mutant had the absorption maximum (λmax) at 487 nm and its spectrum was also shifted into the UV region to form meta II by yellow light irradiation. During subsequent incubation in the dark, decreased absorbance in the UV region and a concomitant increase of the absorbance at around 485 nm were observed (Figure 1F). Analysis of the retinal configurations showed that light irradiation triggered the isomerization of the retinal to the all-trans form, which converted back to the 11-cis form during the subsequent incubation in the dark (Figure 1H). This interconversion of the retinal isomers can explain the spectral change of G188C/N2C/D282C mutant after light irradiation. Thermal recovery of the original dark state after light irradiation was also observed at 37°C (Figure 1G). In addition, G188C mutant showed thermal recovery of the absorption spectrum of the original dark state after light irradiation at 20°C (Figure 1—figure supplement 2B), which was confirmed by an increase of the amount of 11-cis retinal during the incubation after light irradiation (Figure 1—figure supplement 2C). Collectively, these results showed that G188C mutation led to acquisition of the ability to thermally recover the original dark state from the photoactivated state.

Figure 1 with 5 supplements see all
Thermal recovery of bovine rhodopsin G188C mutant after yellow light irradiation.

Thermal stability of wild-type (A) and G188C (B) and G188C/N2C/D282C (C) mutants of bovine rhodopsin purified after the incubation with 11-cis retinal. Absorption spectra were recorded after 0, 5, 10, 15, and 20 min incubation (curves 1–5, respectively) in the dark at 37°C. (D) The schematic presentation of the retinal configuration change of wild-type bovine rhodopsin. The dark state, meta II, and meta III contain 11-cis-15-anti retinal, all-trans-15-anti retinal and all-trans-15-syn retinal, respectively (Ritter et al., 2008). Absorption spectra of N2C/D282C (E) and G188C/N2C/D282C (F) mutants of bovine rhodopsin purified after the incubation with 11-cis retinal. Spectra were recorded in the dark (curve 1) and 0, 5, 15, 30, 60, and 120 min after yellow light irradiation (curves 2–7, respectively) at 20°C. (Inset) Difference spectra obtained by subtracting the spectrum just after irradiation (curve 2 in (E) and (F)) from the spectra measured after irradiation (curves 3–7 in (E) and (F)) (curves 1–5, respectively). (G) Absorption spectra of G188C/N2C/D282C mutant measured at 37°C. Spectra were recorded in the dark (curve 1) and 0.1, 10, 50, 100, and 1000s after yellow flash light irradiation (curves 2–6, respectively). (Inset) Difference spectra obtained by subtracting the spectrum just after irradiation (curve 2 in (G)) from the spectra measured after irradiation (curves 3–6 in (G)) (curves 1–4, respectively). (H) Isomeric compositions of retinal of G188C/N2C/D282C mutant. The retinal configurations were analyzed by high-performance liquid chromatography (HPLC) after extraction of the chromophore from the samples before light irradiation and 0, 5, and 60 min after yellow light irradiation at 20°C as shown in Figure 1—figure supplement 4.

We also analyzed whether other G188 mutants acquire the photocyclic property. A previous study showed that G188E and G188R mutants of human rhodopsin cannot form the photopigments after reconstitution with 11-cis retinal (Sung et al., 1993). Thus, we introduced 16 other mutations at position 188 of bovine rhodopsin and prepared the mutant proteins purified after reconstitution with 11-cis retinal. We successfully detected the photopigments from eight of these mutants (curve 1 in Figure 1—figure supplement 3). λmax of the mutants was blue-shifted from that of wild-type (500 nm) with one exception, G188D (509 nm) (Table 1). Yellow light irradiation of these mutants shifted the spectra into the UV region to form meta II (curve 2 in Figure 1—figure supplement 3). During subsequent incubation in the dark at 20°C, each mutant showed characteristic spectral changes (curves 3–8 in Figure 1—figure supplement 3). However, these spectral changes were different from a substantial increase of the absorbance at around their λmax. These results showed that the thermal recovery to the original dark state after light irradiation was not clearly detected in these mutants. Thus, we concluded that the photocyclic property was observed uniquely in G188C mutant.

Table 1
Comparison of λmax in the dark state and spectral components after UV light irradiation.
λmax*Dark state (%)Meta II (%)Meta III (%)
Wild-type50021.613.764.7
G188C48741.248.510.3
G188A49418.151.730.2
G188D50930.967.71.4
G188M4919.210.080.8
G188N49200100
G188Q4932.45.592.1
G188S49514.637.747.7
G188T48811.02.986.1
G188V4867.363.429.3
  1. *

    λmax was estimated from the absorption spectra shown in Figure 2 and Figure 2—figure supplement 1.

  2. Component ratios of the dark state, meta II, and meta III were calculated based on the spectral changes induced by UV light irradiation shown in Figure 2 and Figure 2—figure supplement 1.

  3. Ratios of the dark state in wild-type and G188C are comparable to those of 11-cis retinal obtained by the retinal configuration analysis (24% in wild-type and 40% in G188C).

Acquisition of photoreversible property of bovine rhodopsin G188C mutant

We also analyzed whether meta II of G188C mutant converts back to the original dark state in a light-dependent manner. We cooled wild-type and G188C mutant to 0°C to prevent the thermal reaction of meta II and measured their spectral changes induced by yellow light and subsequent UV light irradiation. Yellow light irradiation of wild-type resulted in the formation of meta II, and subsequent UV light irradiation shifted the spectrum into the visible region with λmax (~470 nm) blue-shifted from that of the original dark state (Figure 2A). Previous studies revealed that this state is comparable to meta III (Figure 1D; Bartl et al., 2001; Ritter et al., 2008). We also constructed template absorption spectra of the dark state, meta II, and meta III modeled by the previous method (Lamb, 1995; Govardovskii et al., 2000; Figure 2—figure supplement 1B) and fitted the difference spectrum (curve 2 in the inset of Figure 2A) calculated by subtracting the spectrum after yellow light irradiation from that after UV light irradiation. Our fitting analysis showed that meta III was formed much more efficiently than the original dark state by UV light irradiation of meta II (Figure 2—figure supplement 1C and Table 1). This spectral analysis was consistent with the observation that UV light irradiation produced a very limited amount of 11-cis retinal from a large amount of all-trans retinal (Figure 2C). These results confirmed that UV light irradiation of meta II induces the syn/anti isomerization of the C = N double bond of the Schiff base more efficiently than it does the cis/trans isomerization of the retinal.

Figure 2 with 2 supplements see all
Photoreaction, retinal configuration, and G protein activation of bovine rhodopsin G188C mutant.

Absorption spectra of wild-type (A) or G188C mutant (B) of bovine rhodopsin purified after the incubation with 11-cis retinal at 0°C. Spectra were recorded in the dark (curve 1), after yellow light (>500 nm) irradiation (curve 2), after subsequent UV light (360 nm) irradiation (curve 3) and after yellow light reirradiation (curve 4). (Inset) Spectral changes of wild-type (A) or G188C mutant (B) induced by yellow light irradiation (curve 1), subsequent UV light (curve 2) irradiation and yellow light reirradiation (curve 3). Difference spectra were calculated based on the spectra shown in (A) and (B). Isomeric compositions of retinal of wild-type (C) and G188C mutant (D). The retinal configurations were analyzed by high-performance liquid chromatography (HPLC) after extraction of the chromophore from the samples before light irradiation, after yellow light irradiation, after subsequent UV light irradiation and after yellow light reirradiation at 0°C as shown in Figure 2—figure supplement 2. (E) Gi-type of G protein activation ability of wild-type. The activation ability was measured in the dark (closed circle) and after yellow light irradiation (open circle). (F) Gi-type of G protein activation ability of G188C mutant. The activation ability was measured in the dark (closed circles), after yellow light irradiation (open circles), after subsequent UV light irradiation (open triangles) and after yellow light reirradiation (open diamonds). Data shown in (E) and (F) were obtained at 0°C and are presented as the means ± SEM of three independent experiments. (G) Absorption spectrum of G188C/N2C/D282C mutant purified after incubation with 11-cis retinal at 0°C. Spectra were recorded in the dark (curve 1), after yellow light (>500 nm) irradiation (curve 2), after subsequent UV light (360 nm) irradiation (curve 3), after yellow light reirradiation (curve 4) and after UV light reirradiation (curve 5). (Inset) Spectral changes induced by yellow light irradiation (curve 1), subsequent UV light (curve 2) irradiation, yellow light reirradiation (curve 3), and UV light reirradiation (curve 4). Difference spectra were calculated based on the spectra shown in (G).

Yellow light irradiation of G188C mutant induced the formation of meta II, and subsequent UV light irradiation shifted the spectrum into the visible region with λmax quite similar to that of the original dark state (Figure 2B). Yellow light reirradiation caused formation of a state whose spectrum almost overlapped with that induced by the first yellow light irradiation (curve 4 in Figure 2B). Spectral changes induced by UV light irradiation and yellow light reirradiation were mirror images of each other (curves 2 and 3 in the inset of Figure 2B). We fitted the UV light-dependent spectral change with the template spectra and showed that the original dark state was formed much more efficiently than meta III by UV light irradiation of meta II (Figure 2—figure supplement 1C and Table 1). This was supported by the observation that UV light irradiation of G188C mutant increased the amount of 11-cis retinal more efficiently than UV light irradiation of wild-type (Figure 2D). These results suggested that meta II of G188C mutant can efficiently photoconvert back to the original dark state. Next, we measured the ability of G188C mutant to activate Gi-type of G protein, because bovine rhodopsin can activate not only transducin but also Gi/Go-types of G protein (Yamashita et al., 2000; Terakita et al., 2002). Our GTPγS-binding assay showed that the light-dependent Gi activation ability of G188C was equivalent to that of wild-type (Figure 2E, F). Subsequent UV light irradiation of G188C mutant suppressed the ability and yellow light reirradiation increased the ability (Figure 2F), which can be explained by the changes of the absorption spectra and the retinal configurations (Figure 2B, D). In addition, G188C/N2C/D282C mutant could also exhibit interconvertibility between the original dark state and meta II upon yellow light and UV light irradiation at 0°C (Figure 2G). These data showed that G188C mutant acquired the property of photoreversibility between the dark state and meta II. We also analyzed the photoreaction of eight other mutants. In all of these mutants, we observed the spectral shift to the UV region by yellow light irradiation and the reincrease of the absorbance in the visible region by subsequent UV light irradiation (Figure 2—figure supplement 1A, C). Fitting of the difference spectra calculated before and after UV light irradiation using template spectra of the dark state, meta II, and meta III (cyan curves in Figure 2—figure supplement 1C) provided information about the component ratios of the dark state, meta II, and meta III after UV light irradiation (Table 1). These results indicated that the recovery to the original dark state upon UV light irradiation occurs most efficiently in G188C mutant.

Speeding up of the photocycle in G188C mutant

Next, we investigated whether the alteration of the lifetime of meta II can modulate the photocycle rate of G188C mutant. It has been reported that E122Q mutation of vertebrate rhodopsin accelerates the decay of meta II and thus shortens the lifetime of meta II (Imai et al., 1997; Imai et al., 2007). Therefore, we prepared E122Q/G188C/N2C/D282C mutant and measured its spectral change after light irradiation. Our spectral and retinal configuration analyses confirmed the thermal recovery of the original dark state in E122Q/G188C/N2C/D282C mutant after light irradiation at 0°C (Figure 3A, B). In addition, the photocycle rate of E122Q/G188C/N2C/D282C mutant at 37°C (Figure 3C) was about 12 times faster than that of G188C/N2C/D282C mutant (Figure 3D). Thus, alteration of the lifetime of meta II by single mutation successfully speeded up the photocycle of G188C mutant.

Figure 3 with 1 supplement see all
Faster recovery rate of the photocycle of bovine rhodopsin G188C mutant by introducing E122Q mutation.

(A) Absorption spectra of E122Q/G188C/N2C/D282C mutant measured at 0°C. Spectra were recorded in the dark (curve 1) and 0, 5, and 60 min after yellow light (>500 nm) irradiation (curves 2–4, respectively). (Inset) Difference spectra obtained by subtracting the spectrum before irradiation (curve 1 in (A)) from the spectra measured after irradiation (curves 2–4 in (A)) (curves 1–3, respectively). (B) Isomeric compositions of retinal of E122Q/G188C/N2C/D282C mutant. The retinal configurations were analyzed by high-performance liquid chromatography (HPLC) after extraction of the chromophore from the samples before light irradiation and 0, 5, and 60 min after yellow light irradiation at 0°C as shown in Figure 3—figure supplement 1. (C) Absorption spectra of E122Q/G188C/N2C/D282C mutant measured at 37°C. Spectra were recorded in the dark (curve 1) and 0.1, 1, 5, 10, and 50 s after yellow flash light irradiation (curves 2–6, respectively). (Inset) Difference spectra obtained by subtracting the spectrum before irradiation (curve 1 in (C)) from the spectra measured after irradiation (curves 2–6 in (C)) (curves 1–5, respectively). (D) Comparison of the thermal recovery process between G188C/N2C/D282C (red) and E122Q/G188C/N2C/D282C (blue). Difference absorbance at λmax obtained by subtracting the spectrum before irradiation from the spectra measured after irradiation shown in Figure 1G and (C) was plotted against time elapsed after irradiation. The time constants of the thermal recovery to the dark state of G188C/N2C/D282C and E122Q/G188C/N2C/D282C mutants at 37°C were 57.4 and 5.1 s, respectively.

Modulation of G protein activation ability by the photocyclic property

We also investigated whether the acquisition of the photocyclic property by G188C mutation affects the G protein activation ability. As shown in Figure 2, light-dependent Gi activation ability was equivalent between wild-type and G188C mutant at 0°C, where substantial thermal recovery to the original dark state was not observed in G188C mutant. Thus, we measured the intracellular cAMP level in cultured cells using a cAMP biosensor (GloSensor) and compared the change of the luminescence from the biosensor triggered by bovine rhodopsin. The increase of the cAMP level induced by the addition of forskolin was attenuated by yellow light irradiation in the N2C/D282C bovine rhodopsin-transfected cells (Figure 4A, B), but not in the mock-transfected cells (Figure 4G), and it subsequently recovered slowly. By contrast, in the G188C/N2C/D282C mutant-transfected cells, we observed rapid recovery of the cAMP level after the decrease of the level induced by yellow light irradiation (Figure 4C, D). In addition, in the E122Q/G188C/N2C/D282C mutant-transfected cells, the cAMP level recovered more quickly from the decrease induced by yellow light irradiation (Figure 4E, F). These results showed that the acquisition of the photocyclic property by G188C mutation changes the G protein activation profile by promoting fast recovery to the original dark state.

Light-mediated suppression of intracellular cAMP level by bovine rhodopsin mutants.

The cAMP levels in N2C/D282C- (A, B), G188C/N2C/D282C- (C, D), E122Q/G188C/N2C/D282C- (E, F), and mock- (G) transfected HEK293T cells were measured using the GloSensor cAMP assay at room temperature. The cells were incubated with 5 µM 11-cis retinal for 2 hr and subsequently treated with 2 µM forskolin prior to exposure to yellow light (>500 nm). Data were normalized to the maximum point before light irradiation. Detailed profiles of the light-dependent cAMP level changes in N2C/D282C, G188C/N2C/D282C, and E122Q/G188C/N2C/D282C are shown in (B), (D), and (F), respectively.

Formation of the photopigments by G188C mutant upon the addition of all-trans retinal

Finally, we analyzed whether G188C mutant forms the photopigments after reconstitution with all-trans retinal. We purified wild-type and G188C mutant after the addition of all-trans retinal to the suspension of rhodopsin-expressing cell membranes. The absorption spectrum of wild-type had almost no peaks in the visible and near-UV regions (Figure 5A). By contrast, the absorption spectrum of G188C mutant had a peak in the visible region (curve 1 in Figure 5B), which was derived from predominant incorporation of 11-cis and 9-cis retinals, not all-trans retinal (Figure 5C). Yellow light irradiation of this pigment resulted in conversion of the retinal to all-trans form to shift the spectrum into the UV region, and subsequent UV light irradiation reincreased the absorbance in the visible region by the isomerization of the retinal from the all-trans to the 11-cis form at 0°C (Figure 5B, C). This was quite similar to the finding for G188C mutant purified after reconstitution with 11-cis retinal (Figure 2B, D).

Figure 5 with 1 supplement see all
Formation of the photopigments of bovine rhodopsin G188C mutant after incubation with all-trans retinal.

Absorption spectra of wild-type (A) or G188C mutant (B) purified after the addition of all-trans retinal to the suspension of rhodopsin-expressing cell membranes at 0°C. Spectra were measured in the dark (curve 1), after yellow light (>500 nm) irradiation (curve 2), after subsequent UV light (360 nm) irradiation (curve 3) and after yellow light reirradiation (curve 4). (Inset) Spectral change caused by yellow light irradiation (curve 1), subsequent UV light irradiation (curve 2) and yellow light reirradiation (curve 3). (C) Isomeric compositions of retinal of G188C mutant purified after the addition of all-trans retinal. The retinal configurations were analyzed by high-performance liquid chromatography (HPLC) after extraction of the chromophore from the samples before light irradiation, after yellow light irradiation, after subsequent UV light irradiation and after yellow light reirradiation shown in Figure 5—figure supplement 1. Regeneration of the photopigments by the addition of 11-cis (D, E) or all-trans (F, G) retinal to purified apo-protein of N2C/D282C. (D) Spectra were measured before (curve 1) and 0, 3, 6, 15, 30, 60, and 120 min after the addition of 1.1 μM 11-cis retinal (curves 2–8). (E) Difference spectra were calculated by subtracting the spectrum just after the addition of 11-cis retinal (curve 2 in (D)) from the spectra measured 3, 6, 15, 30, 60, and 120 min after the addition of 11-cis retinal (curves 3–8 in (D)) (curves 1–6, respectively). (F) Spectra were measured before (curve 1) and 0, 0.5, 1, 2, 6, 12, and 16 hr after the addition of 1.1 μM all-trans retinal (curves 2–8). (G) Difference spectra were calculated by subtracting the spectrum just after the addition of all-trans retinal (curve 2 in (F)) from the spectra measured 0.5, 1, 2, 6, 12, and 16 hr after the addition of all-trans retinal (curves 3–8 in (F)) (curves 1–6, respectively). Regeneration of the photopigments by the addition of 11-cis (H, I) or all-trans (J, K) retinal to purified apo-protein of G188C/N2C/D282C. (H) Spectra were measured before (curve 1) and 0, 3, 6, 15, 30, 60, and 120 min after the addition of 1.1 μM 11-cis retinal (curves 2–8). (I) Difference spectra were calculated by subtracting the spectrum just after the addition of 11-cis retinal (curve 2 in (H)) from the spectra measured 3, 6, 15, 30, 60, and 120 min after the addition of 11-cis retinal (curves 3–8 in (H)) (curves 1–6, respectively). (J) Spectra were measured before (curve 1) and 0, 0.5, 1, 2, 6, 12, and 16 hr after the addition of 1.1 μM all-trans retinal (curves 2–8). (K) Difference spectra were calculated by subtracting the spectrum just after the addition of all-trans retinal (curve 2 in (J)) from the spectra measured 0.5, 1, 2, 6, 12, and 16 hr after the addition of all-trans retinal (curves 3–8 in (J)) (curves 1–6, respectively). (L) Regeneration processes of the photopigments of N2C/D282C (red curve) and G188C/N2C/D282C (blue curve) by the addition of all-trans retinal as shown in (F) and (J) were monitored by the change of absorbance at 500 nm.

We also prepared the purified apo-proteins of N2C/D282C and G188C/N2C/D282C and investigated the process of regeneration of the photopigments upon the addition of 11-cis or all-trans retinal. The addition of 11-cis retinal to N2C/D282C and G188C/N2C/D282C quickly increased the absorbance at around 505 nm (Figure 5D, E) and 490 nm (Figure 5H, I), respectively, which showed the formation of their 11-cis retinal bound dark states. The addition of all-trans retinal to N2C/D282C resulted in a slight increase of the absorbance at around 480 nm (Figure 5F, G), whereas the addition of all-trans retinal to G188C/N2C/D282C resulted in a substantial increase of the absorbance at around 485 nm (Figure 5J, K). The regeneration ability of G188C/N2C/D282C in response to the addition of all-trans retinal was much higher than that of N2C/D282C (Figure 5L). These results showed that G188C mutant can uniquely form the photopigments upon the addition of not only 11-cis retinal but also all-trans retinal.

Mechanism of conversion of the molecular property by G188C mutation

In general, vertebrate rhodopsin photoconverts to a metastable active state, meta II, by the cis/trans isomerization of the retinal and subsequently undergoes a thermal transition to meta III by the syn/anti isomerization of the C = N double bond of the Schiff base (Figure 1D; Ritter et al., 2008; Vogel et al., 2003; Hofmann et al., 2009). Thus, in response to light and heat, meta II exhibits greater conversion from all-trans-15-anti to all-trans-15-syn retinal than from all-trans to 11-cis retinal and recovers to the original dark state very inefficiently. By contrast, G188C mutant can revert to the original dark state from meta II much more efficiently than wild-type, possibly as a result of the preferential isomerization from all-trans to 11-cis retinal within the chromophore-binding pocket of meta II. The molecular models of the dark state and meta II of G188C mutant constructed based on the crystal structures of wild-type (Choe et al., 2011; Okada et al., 2004) suggest that Cys188 can be located in the vicinity of the C11 = C12 position of the retinal and Glu181 (Figure 1—figure supplement 1B). The protonation of the Schiff base in the dark state is neutralized by Glu113 (Sakmar et al., 1989; Zhukovsky and Oprian, 1989; Nathans, 1990), from which the counterion position switches to Glu181 in meta I (Yan et al., 2003; Lüdeke et al., 2005), a precursor of meta II. It should be noted that G188D mutant formed a substantial amount of meta I after light irradiation (Figure 2—figure supplement 1A), which can be explained by the stabilization of the protonated Schiff base of meta I by the introduction of an aspartic acid residue. Moreover, during the process of the formation of meta II, Cys188, Glu181, and the adjacent water molecule approach the Schiff base, whereas Glu113 and Ser186 move away from the Schiff base (Figure 1—figure supplement 1B). A previous study suggested that the syn/anti isomerization of the C = N double bond of the Schiff base in meta II can be promoted by the appropriate hydrogen-bonding network around the Schiff base as well as the specific steric chromophore–protein interaction (Vogel et al., 2003). Thus, we speculate that the cysteine residue introduced at position 188 disturbs the local structure and the hydrogen-bonding network around the Schiff base and Glu181 in meta II, which prevents the syn/anti isomerization of the C = N double bond of the Schiff base.

The addition of all-trans retinal to G188C mutant resulted in the formation of 9-cis or 11-cis retinal-containing photopigments (Figure 5). 9-cis or 11-cis retinal within G188C mutant would be formed from all-trans retinal on the outside or inside of the protein. If the thermal isomerization of the retinal occurs on the outside of the protein, we can expect the formation of the photopigments also from wild-type. However, this occurred much less efficiently in wild-type. Thus, we speculate that 9-cis or 11-cis retinal would be formed from all-trans retinal on the inside of G188C mutant, which is consistent with the acceleration of the thermal cis/trans isomerization of the retinal in meta II of G188C mutant.

Quite recently, we showed that T188C mutation of a bistable opsin, Opn5m, induces the thermal isomerization of the retinal from cis to trans isomer to make the opsin photocyclic (Fujiyabu et al., 2022). In this study, we observed the thermal recovery from the photoactivated state in G188C mutant (Figure 1 and Figure 1—figure supplement 2), but not in other mutants (Figure 1—figure supplement 3). Thus, the cysteine residue at position 188 would have a special role to facilitate the thermal isomerization of the retinal. In a previous study of Opn5L1, we revealed that the light-dependent adduct formation between Cys188 and 11-cis retinal accelerates the isomerization to all-trans retinal to recover the original dark state (Sato et al., 2018). During this process of photocycling of Opn5L1, our spectral analysis detected an increase of the absorbance at around 270 nm (Figure 1—figure supplement 5A, B), which is derived from the breaking of the retinal-conjugated double bond system by the adduct formation. However, in this study, the introduction of the cysteine residue at position 188 of bovine rhodopsin induced the isomerization from all-trans to 11-cis retinal, which is a reverse reaction to the thermal isomerization of the retinal in Opn5L1. Moreover, we could not observe a clear increase of the absorbance at around 270 nm during the thermal reaction of bovine rhodopsin G188C mutant after photoreception (Figure 1—figure supplement 5C, D). Thus, the detailed molecular mechanism of the acceleration of the thermal recovery to the original dark state in G188C mutant remains unknown, but one possible mechanism is that the cysteine residue introduced at position 188 transiently forms an adduct with all-trans retinal after photoactivation and quickly dissociates from the retinal after the isomerization to 11-cis retinal. An alternative possible mechanism can be predicted based on the analysis of channelrhodopsins. Channelrhodopsins have a unique cysteine residue near the C13 position of the retinal, which modulates the photocycle rate by regulating the syn/anti isomerization of the C = N double bond of the Schiff base without the adduct formation (Ritter et al., 2013; Oppermann et al., 2019). Considering this molecular mechanism in channelrhodopsins, the cysteine residue at position 188 near the C11 = C12 position of the retinal may possibly influence the structure and charge distribution of the retinal in meta II, which could trigger the thermal cis/trans isomerization of the retinal.

Comparison of the amino acid sequences reveals that most bistable opsins have a threonine or serine residue at position 188 (Figure 1—figure supplement 1A). Thus, we speculate that an ancestral bistable opsin possessed a threonine or serine residue at position 188, which was mutated into the glycine residue in the common ancestor of monostable opsins, including vertebrate rhodopsin, cone visual pigments, and pinopsin. Also, the mutation at position 188 may have contributed to the change from the bistable property to the monostable property in the evolutionary ancestor of vertebrate rhodopsin. This is consistent with our recent finding that mutations at Thr188 of a bistable opsin, Opn5m, drastically hamper the bistable photoreaction (Fujiyabu et al., 2022). However, we could not create a bistable opsin by a single mutation, G188T or G188S, of bovine rhodopsin (Figure 2—figure supplement 1 and Table 1), which means that additional amino acid residue(s) are necessary to explain the difference between the bistable and monostable property from the viewpoint of the molecular evolution of opsins.

Conclusion

In this study, we analyzed a series of mutants at position 188 of bovine rhodopsin and found that G188C mutant has a unique active state which can revert to the original dark state both by a thermal reaction and by a photoreaction. These results showed that the molecular properties of vertebrate rhodopsin can be converted to photocyclic and photoreversible properties by this single mutation. Little attention has been paid to the functional role of the residue at position 188 in opsins so far. The combination of the mutation at position 188 with other mutations in various opsins could perturb the local structure around the Schiff base, which could lead to interconversion of the molecular properties among opsins. Moreover, G188C mutant of bovine rhodopsin has several advantages as an optogenetic tool, because G188C mutant can be reconstituted in the presence of all-trans retinal and exhibits the photocyclic property, like channelrhodopsin (Deisseroth and Hegemann, 2017) in addition to its high expression yield in mammalian cultured cells and high G protein activation ability. We successfully showed that the change of the lifetime of meta II by the single mutation can modulate the photocycle rate of G188C mutant. Further accumulation of evidence about mutants of vertebrate rhodopsin can help to guide the modification of the molecular properties of G188C mutant, which will provide a novel type of optogenetic tools based on vertebrate rhodopsin.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Commercial assay or kitIn-Fusion HD CloningClontechClontech: 639,647
Commercial assay or kitGloSensor cAMP AssayKitPromegaPromega: E1290
Chemical compound, drugForskolinFUJIFILM (Wako)FUJIFILM:067-02191
Chemical compound, drugn-Dodecyl-β-D-maltosideDOJINDODOJINDO: D316
Chemical compound, drug[35S]GTPγSPerkinElmerPerkinElmer: NEG030H
Software, algorithmIgor Pro Ver. 6https://www.wavemetrics.com/
Software, algorithmPyMOL Ver. 1.1https://pymol.org/2/

Preparation of bovine rhodopsin mutants

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The mutant cDNAs of bovine rhodopsin (accession no. AB062417) were constructed using an In-Fusion cloning kit (Clontech). The wild-type and mutant cDNAs of bovine rhodopsin were inserted into the mammalian expression vector pUSRα (Kayada et al., 1995) or pCAGGS (Niwa et al., 1991). HEK293T cells were kindly provided by Dr. Satoshi Koike (Tokyo Metropolitan Institute of Medical Science, Tokyo Japan) (Onishi et al., 1999) and were authenticated by short tandem repeat profiling. The cells tested negative for mycoplasma contamination. The plasmid was transfected into HEK293T cells using the calcium-phosphate method. After 2-day incubation, the transfected cells were collected by centrifugation and suspended in Buffer A (50 mM HEPES, 140 mM NaCl, 3 mM MgCl2, pH 6.5), and 11-cis or all-trans retinal was added to the cell suspension to reconstitute the photopigments. They were solubilized in Buffer A containing 1% dodecyl maltoside (DDM) and adsorbed to a Rho1D4 (anti-bovine rhodopsin monoclonal antibody) affinity column to purify the pigments. After washing the column with Buffer A containing 0.02% DDM, the pigment was eluted by adding synthetic peptide with the epitope sequence. To purify the apo-proteins of rhodopsin, the transfected cell membranes without the addition of retinal were solubilized in Buffer A containing 1% DDM and adsorbed to a Rho1D4 affinity column.

Spectroscopic measurements

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UV/Vis absorption spectra were recorded with a UV–visible spectrophotometer (UV-2450 and UV-2400, Shimadzu). Samples were kept at 0, 20, or 37°C using a cell holder equipped with a temperature-controlled circulating water bath in order to analyze the thermal reaction of the pigments in detail. The samples were irradiated with either yellow light through a Y-52 cutoff filter (Toshiba) or UV light through a UVD-36 glass filter (AGC Techno Glass) from a 1 kW tungsten halogen lamp (Master HILUX-HR; Rikagaku).

To monitor the process of the photocycle of G188C mutant of bovine rhodopsin, a time-resolved CCD spectrophotometer (C10000 system, Hamamatsu Photonics) was used (Sakai et al., 2012). Spectra were taken from G188C mutant samples in the dark and at different time points after irradiation (170-μs, yellow light through a Y-52 cutoff filter from a Xenon flash lamp). The temperature of the sample was kept at 37°C by a temperature controller (pqod, QUANTUM Northwest). Absorbance changes at λmax were plotted as a function of time and fitted with a single-exponential function to obtain the time constants for the recovery to the original dark state.

Retinal configuration analysis

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Retinal configurations within rhodopsin samples were analyzed by high-performance liquid chromatography (LC-10ATvp; Shimadzu) with a silica column (YMC-Pack SIL, particle size 3 μm, 150 × 6.0 mm, YMC) as previously described (Tsutsui et al., 2007).

G protein activation assay

The activation of Gi-type of G protein was measured by GDP/GTPγS exchange of G protein using a radionucleotide filter-binding assay (Yamashita et al., 2000; Yamashita et al., 2010). Giαβγ was prepared by mixing rat Giα1 expressed in Escherichia coli strain BL21 (Lee et al., 1994) with Gtβγ purified from bovine retina (Tachibanaki et al., 1997). All of the assay procedures were carried out at 0°C. The assay mixture consisted of 10 nM pigment, 600 nM G protein, 50 mM HEPES (pH 7.0), 140 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.01% DDM, 1 μM [35S]GTPγS, and 2 μM GDP. Bovine rhodopsin wild-type and G188C mutant purified after reconstitution with 11-cis retinal were mixed with G protein solution and were kept in the dark or irradiated with yellow light (>500 nm) for 1 min, with subsequent UV light for 1 min or with yellow light reirradiation for 1 min. After irradiation, the GDP/GTPγS exchange reaction was initiated by the addition of [35S]GTPγS solution to the mixture of rhodopsin and G protein. After incubation for the selected time in the dark, an aliquot (20 μl) was removed from the sample into 200 μl of stop solution (20 mM Tris/Cl [pH 7.4], 100 mM NaCl, 25 mM MgCl2, 1 μM GTPγS, and 2 μM GDP), and it was immediately filtered through a nitrocellulose membrane to trap [35S]GTPγS bound to G proteins. The amount of bound [35S]GTPγS was quantitated by assaying the membrane with a liquid scintillation counter (Tri-Carb 2910 TR; PerkinElmer).

cAMP level measurement in cultured cells

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cAMP levels in HEK293T cells were measured using the GloSensor cAMP assay (Promega) according to the manufacturer’s instructions and a previous report (Bailes and Lucas, 2013). HEK293T cells were seeded in 96-well plates at a density of 20,000 cells/well in low serum medium (D-MEM/F12 containing 0.25% FBS). After 24 hr of incubation, cells were transfected with 50 ng of rhodopsin plasmid and 50 ng of Glosensor 22F plasmid per well by the polyethylenimine transfection method. After overnight incubation, the medium was replaced with an equilibration medium which contained a 2% dilution of the GloSensor cAMP reagent stock solution, 10% FBS and 5 µM retinal in a CO2-independent medium (Thermo Fisher Scientific). Following 2-hr equilibration at room temperature, luminescence from the cells was measured using a microplate reader (SpectraMax L, Molecular Devices). For the measurement of Gi activation by wild-type and mutant rhodopsin, the cells were first treated with 2 µM forskolin to increase the cAMP-dependent luminescence to the plateau level and subsequently stimulated for 30 s with yellow light through a Y-52 cutoff filter from a 1 kW tungsten halogen lamp.

Data availability

All data needed to evaluate the conclusions are present in the paper. The datasets of the current study are available in the Dryad repository (https://doi.org/10.5061/dryad.c866t1g88).

The following data sets were generated
    1. Yamashita T
    (2022) Dryad Digital Repository
    Data from: Creation of photocyclic vertebrate rhodopsin by single amino acid substitution.
    https://doi.org/10.5061/dryad.c866t1g88

References

    1. Kayada S
    2. Hisatomi O
    3. Tokunaga F
    (1995) Cloning and expression of frog rhodopsin cDNA
    Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology 110:599–604.
    https://doi.org/10.1016/0305-0491(94)00179-x
    1. Shichida Y
    2. Matsuyama T
    (2009) Evolution of opsins and phototransduction
    Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 364:2881–2895.
    https://doi.org/10.1098/rstb.2009.0051
    1. Sung CH
    2. Davenport CM
    3. Nathans J
    (1993)
    Rhodopsin mutations responsible for autosomal dominant retinitis pigmentosa Clustering of functional classes along the polypeptide chain
    The Journal of Biological Chemistry 268:26645–26649.

Decision letter

  1. Andrew C Kruse
    Reviewing Editor; Harvard Medical School, United States
  2. Richard W Aldrich
    Senior Editor; The University of Texas at Austin, United States
  3. Peter Hegemann
    Reviewer; Humboldt University of Berlin, Germany
  4. Gebhard Schertler
    Reviewer; Paul Scherrer Institute, Switzerland

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Creation of photocyclic vertebrate rhodopsin by single amino acid substitution" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Aldrich as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Peter Hegemann (Reviewer #1); Gebhard Schertler (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

Please see the discussion below regarding clarity and presentation of data. While no new experiments are necessary, the reviewers felt that improvements to the text would significantly enhance readability and breadth of appeal.

Reviewer #1 (Recommendations for the authors):

The manuscript of Sakai et al., reports about the long-standing question what internal constrains discriminate a cyclic rhodopsin kinetics from non-cyclic kinetics. This question has been discussed over many decades of rhodopsin research.

Based on the authors report about chicken OPN5L1 (Ref.7), which forms a thioadduct between a reactive Cys and the retinal upon illumination, they introduced such a Cys into the homologous position of bovine rhodopsin, G188C, rendering the naturally non-cyclic photoreaction cyclic. The photoproduct Meta II does not release the at-retinal and the rhodopsin converts back to the dark state. Other residues at this position were only partially active or unstable. At low temperature Meta II is further stabilized but may be photoconverted back to the dark state. This sounds trivial but it is not because wt rhodopsin Meta II state photoconverts it to Meta III with 15=N syn geometry thermally or photochemically and does not recover to the dark state.

The authors further stabilized the G188C-photoproducts by introducing two more Cys as reported already by Dan Oprian et al., in 2003. The triple mutant showed a further stabilized Meta II photoproduct whereas additional E122Q mutation reaccelerated it.

Finally, the authors showed that G188C mutants can also be reconstituted with at-retinal which is a great advantage over wt for potential optogenetic applications.

The G188C variants are fully active in terms of G-protein binding and the cAMP assay revealed the thermal photocycle kinetics of wt and mutants (Figure 4).

Comments:

What the reader also need to show are the differences between dark state and Meta II which could be visualized easily by an overlay of the two available structures.

The main question for the reader is to what extent can the principle of cyclization by introducing a Cys homologous positions be extended to other vertebrate rhodopsins. If this would be the case it would be a spectacular observation and open a new field for application. Now, it still looks exotic and as a exceptional observation.

The spectra of G188C at 37degC are of poor quality compared to the double or triple mutants in Figure 1 or the G188C spectrum in Figure S2. I would show the 20degC spectra in Figure 1.

The non-specialized reader would appreciate a scheme of cis-trans and "syn/anti" isomerization and if I interpret Figure S7 correctly is should be an anti/syn illumination because dark state and Meta II should be anti? Please clarify.

Figure S7 is helpful but should be introduced earlier in the text to make clear where C188 is located. I really do expect a deeper discussion of this issues including other relevant residues in Figure S7.

Interpretation

The weaker part of the manuscript is the interpretation of why G188C mutant prevents retinal release during the Meta II state and its thermal or photochemical anti/syn isomerization. Many invertebrate rhodopsins do not release the retinal during the Meta II state and do not have any Cys residue at this location. Moreover, some of them deprotonate during the meta state and others do not. The authors are experts in invertebrate rhodopsins as well and privileged to give a better interpretations.

Next, the suggestion that Cys causes transient thioadduct formation during the photocycle is not justified by anything and should be proposed with more care. For example a Cys at a similar position has been found in microbial Channelrhodopsins (ChRs). Substitution of this Cys slows down the photocycle dramatically and prevents syn/anti isomerization as it occurs during light adaptation. On the other hand some rhodopsin do not show such an anti/syn isomerization at all but removal of the Cys near the C13 position promotes anti/syn isomerization as found for example in Mermaid ChRs (Oppermann et al.,). The sulfur of the Cys seems to act as a nucleophile for retinal polyene chain, forming a thioadduct as in OPN5L or not as in Channelrhodopsin. But the Cys obviously influences the charge distribution in darkness or during the excited state. Moreover, residues in the active site including E113, E188 and other residues could act as steric constraints that influence the isomerization specificity as well.

I really do expect deeper discussion of this issues including other relevant residues in Figure S7.

What the reader also needs to know are the differences between dark state and Meta II which could be visualized by an overlay of the two available structures.

Reviewer #2 (Recommendations for the authors):

I support the publication without further modifications.

Reviewer #3 (Recommendations for the authors):

The data are thorough and convincing. However, the quality of presentation must be significantly improved.

1. Overall the manuscript is too long, with numerous unnecessary repetitions. It can be shortened by 30-40% without losing the message.

2. Evolutionary analysis (Figure S1) suggests that Ala, Ser, or Thr are in 188 position of most bistable opsins. How do the authors interpret this in the context of their data? The authors should discuss the difference in the properties of vertebrate opsin with Ala, Ser, or Thr are in 188 position (Figure S4), as compared to 188Cys? These data suggest that the residue in position 188 is not the only determinant of rhodopsin properties. The authors might want to revise Figure S7 accordingly.

3. Extensive editing, preferably by a native speaker, is needed. Some examples: line 35, "photoreceptions" should be "photoreception"; line 36, "sequences" should be "sequence"; line 50, "reversible with each other by light irradiations" should be "interconvertible by light"; lines 73, 86, 128, 148, 209, 223, delete "or not"; lines 73-74, "change the molecular property of bovine rhodopsin to acquire the photocyclic property" should be "can make bovine rhodopsin photocyclic"; line 91, "condition" should be "conditions"; line 125, "Altogether" should be "Collectively"; lines 152, 195 "irradiations" should be "irradiation"; lines 174-175, "overlapped that" should be "overlapped with that"; etc.

https://doi.org/10.7554/eLife.75979.sa1

Author response

Reviewer #1 (Recommendations for the authors):

[…]

Comments:

What the reader also need to show are the differences between dark state and Meta II which could be visualized easily by an overlay of the two available structures.

According to your comment, we constructed the structural models of the dark state and meta II of G188C mutant based on the 3D structures of the dark state and meta II of wild-type and superimposed these models to visualize the structural difference around position 188. We showed these structural models in Figure 1—figure supplement 1B.

The main question for the reader is to what extent can the principle of cyclization by introducing a Cys homologous positions be extended to other vertebrate rhodopsins. If this would be the case it would be a spectacular observation and open a new field for application. Now, it still looks exotic and as a exceptional observation.

I thank you very much for this important comment. Quite recently, we showed that a vertebrate non-visual bistable opsin, Opn5m, acquired the photocyclic property by the introduction of a cysteine residue at position 188 (Fujiyabu et al., (2022) Commun. Biol. 5, 63). Thus, we think that several opsins, including vertebrate rhodopsin and Opn5m, have the potential to convert into the photocyclic opsin by the introduction of a cysteine residue at position 188. Now we are systematically analyzing which opsins can acquire the photocyclic property by the introduction of a cysteine residue at position 188. To show our recent results, we added the following sentence in the “Results and discussion” section.

Page 13: “Quite recently, we showed that T188C mutation of a bistable opsin, Opn5m, induces the thermal isomerization of the retinal from cis to trans isomer to make the opsin photocyclic.”

The spectra of G188C at 37degC are of poor quality compared to the double or triple mutants in Figure 1 or the G188C spectrum in Figure S2. I would show the 20degC spectra in Figure 1.

The poor quality of the spectra of triple mutant (G188C/N2C/D282C) recorded at 37degC (Figure 1G) is due to the spectral measurement using a CCD spectrophotometer, in which we weakened the intensity of the monitoring light to prevent the rhodopsin bleaching in the sample. We already showed the spectra of the triple mutant at 20degC in Figure 1F, which were recorded by a scanning spectrophotometer with a single monochromator. We would like to show the spectra at 37degC in Figure 1G to confirm that the photocyclic reaction of G188C mutant can be observed at 37degC.

The non-specialized reader would appreciate a scheme of cis-trans and "syn/anti" isomerization and if I interpret Figure S7 correctly is should be an anti/syn illumination because dark state and Meta II should be anti? Please clarify.

According to your comment, we added a new figure (Figure 1D) to clarify the retinal configuration change of wild-type bovine rhodopsin.

Figure S7 is helpful but should be introduced earlier in the text to make clear where C188 is located. I really do expect a deeper discussion of this issues including other relevant residues in Figure S7.

Interpretation

The weaker part of the manuscript is the interpretation of why G188C mutant prevents retinal release during the Meta II state and its thermal or photochemical anti/syn isomerization. Many invertebrate rhodopsins do not release the retinal during the Meta II state and do not have any Cys residue at this location. Moreover, some of them deprotonate during the meta state and others do not. The authors are experts in invertebrate rhodopsins as well and privileged to give a better interpretations.

Next, the suggestion that Cys causes transient thioadduct formation during the photocycle is not justified by anything and should be proposed with more care. For example a Cys at a similar position has been found in microbial Channelrhodopsins (ChRs). Substitution of this Cys slows down the photocycle dramatically and prevents syn/anti isomerization as it occurs during light adaptation. On the other hand some rhodopsin do not show such an anti/syn isomerization at all but removal of the Cys near the C13 position promotes anti/syn isomerization as found for example in Mermaid ChRs (Oppermann et al.,). The sulfur of the Cys seems to act as a nucleophile for retinal polyene chain, forming a thioadduct as in OPN5L or not as in Channelrhodopsin. But the Cys obviously influences the charge distribution in darkness or during the excited state. Moreover, residues in the active site including E113, E188 and other residues could act as steric constraints that influence the isomerization specificity as well.

I really do expect deeper discussion of this issues including other relevant residues in Figure S7.

I thank you very much for the valuable comment about the molecular mechanism underlying the photocyclic reaction in G188C mutant. The crystal structures of the dark state and meta II of bovine rhodopsin suggest that the appropriate hydrogen-bonding network and the specific steric interactions around the Schiff base regulate the deprotonation/reprotonation of the Schiff base and the syn/anti isomerization of the C=N double bond of the Schiff base during the formation and the decay of meta II. However, in the present situation, it is very difficult to clearly construct a molecular model to explain the acceleration of the syn/anti isomerization of the C=N double bond in meta II. In this study, we can speculate that the introduction of a cysteine residue at position 188 would disturb the local environment around the Schiff base and Glu181 in meta II, which prevents the syn/anti isomerization of the C=N double bond. However, in the previous version of the manuscript, a lack of detailed structural information about G188C mutant caused us to hesitate to speculate about the molecular mechanism underlying the photocyclic reaction in G188C mutant except for the acquisition of the special structural mechanism, the adduct formation. During the revision process, you kindly provided valuable information about channelrhodopsins indicating that a unique cysteine residue is located near the C13 position of the retinal and modulates the photocycle rate by regulating the syn/anti isomerization of the C=N double bond of the Schiff base. Thus, based on the comparison of the predicted structures of the dark state and meta II of G188C and the mutational analysis of the cysteine residue of channelrhodopsins, we revised the sentences in the “Results and discussion” section as follows.

What the reader also needs to know are the differences between dark state and Meta II which could be visualized by an overlay of the two available structures.

According to your comment, we constructed the structural models of the dark state and meta II of G188C mutant based on the 3D structures of the dark state and meta II of wild-type and superimposed these models to visualize the structural differences around position 188. We showed these structural models in Figure 1—figure supplement 1B.

Reviewer #2 (Recommendations for the authors):

I support the publication without further modifications.

Thank you very much for your critical reading of our manuscript and for your valuable comments. I am very happy to know that you support the publication of the manuscript without further modifications.

Reviewer #3 (Recommendations for the authors):

The data are thorough and convincing. However, the quality of presentation must be significantly improved.

1. Overall the manuscript is too long, with numerous unnecessary repetitions. It can be shortened by 30-40% without losing the message.

According to your comment, we omitted a number of sentences, leaving the necessary explanations to help the understanding of non-specialist readers.

2. Evolutionary analysis (Figure S1) suggests that Ala, Ser, or Thr are in 188 position of most bistable opsins. How do the authors interpret this in the context of their data? The authors should discuss the difference in the properties of vertebrate opsin with Ala, Ser, or Thr are in 188 position (Figure S4), as compared to 188Cys? These data suggest that the residue in position 188 is not the only determinant of rhodopsin properties. The authors might want to revise Figure S7 accordingly.

Thank you very much for the important comment. Comparison of the amino acid sequences reveals that most bistable opsins have a threonine or serine residue at position 188 (Figure 1—figure supplement 1A). Thus, we speculate that an ancestral bistable opsin possessed a threonine or serine residue at position 188, which was mutated into the glycine residue in the common ancestor of mono-stable opsins. And the mutation at position 188 may have contributed to the loss of the bistable property and the acquisition of the mono-stable property of vertebrate rhodopsin. This is consistent with our recent finding that mutations at Thr188 of a bistable opsin, Opn5m, drastically hamper the bistable photoreaction (Fujiyabu et al., (2022) Commun. Biol. 5, 63). However, we could not create a bistable opsin by G188T or G188S mutation of bovine rhodopsin (Figure 2—figure supplement 1), which means that additional amino acid residues are necessary to explain the difference between the bistable and mono-stable property. To clarify this point, we added the following sentences in the “Results and discussion” section.

Page 14: “Comparison of the amino acid sequences reveals that most bistable opsins have a threonine or serine residue at position 188 (Figure 1—figure supplement 1A). Thus, we speculate that an ancestral bistable opsin possessed a threonine or serine residue at position 188, which was mutated into the glycine residue in the common ancestor of mono-stable opsins, including vertebrate rhodopsin, cone visual pigments and pinopsin. Also, the mutation at position 188 may have contributed to the change from the bistable property to the mono-stable property in the evolutionary ancestor of vertebrate rhodopsin. This is consistent with our recent finding that mutations at Thr188 of a bistable opsin, Opn5m, drastically hamper the bistable photoreaction. However, we could not create a bistable opsin by a single mutation, G188T or G188S, of bovine rhodopsin (Figure 2—figure supplement 1 and Table 1), which means that additional amino acid residue(s) are necessary to explain the difference between the bistable and mono-stable property from the viewpoint of the molecular evolution of opsins.”

3. Extensive editing, preferably by a native speaker, is needed. Some examples: line 35, "photoreceptions" should be "photoreception"; line 36, "sequences" should be "sequence"; line 50, "reversible with each other by light irradiations" should be "interconvertible by light"; lines 73, 86, 128, 148, 209, 223, delete "or not"; lines 73-74, "change the molecular property of bovine rhodopsin to acquire the photocyclic property" should be "can make bovine rhodopsin photocyclic"; line 91, "condition" should be "conditions"; line 125, "Altogether" should be "Collectively"; lines 152, 195 "irradiations" should be "irradiation"; lines 174-175, "overlapped that" should be "overlapped with that"; etc.

I thank you very much for your careful reading of the manuscript. We revised the manuscript according to all of your comments. Also, we tried to improve the English of our manuscript according to the advice from a native speaker of English.

https://doi.org/10.7554/eLife.75979.sa2

Article and author information

Author details

  1. Kazumi Sakai

    Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6631-8546
  2. Yoshinori Shichida

    1. Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan
    2. Research Organization for Science and technology, Ritsumeikan University, Kusatsu, Japan
    Contribution
    Conceptualization, Formal analysis, Funding acquisition, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
  3. Yasushi Imamoto

    Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan
    Contribution
    Funding acquisition, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0803-4163
  4. Takahiro Yamashita

    Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Supervision, Writing – original draft, Writing – review and editing
    For correspondence
    yamashita.takahiro.4z@kyoto-u.ac.jp
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7956-9288

Funding

Ministry of Education, Culture, Sports, Science and Technology (16H02515)

  • Yoshinori Shichida

Ministry of Education, Culture, Sports, Science and Technology (19K21848)

  • Yasushi Imamoto

Ministry of Education, Culture, Sports, Science and Technology (16K07437)

  • Takahiro Yamashita

Japan Science and Technology Agency (JPMJCR1753)

  • Takahiro Yamashita

Kyoto University Education and Research Foundation

  • Takahiro Yamashita

Takeda Science Foundation

  • Takahiro Yamashita

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Dr. Elizabeth Nakajima for a critical reading of the manuscript. We also thank Prof. Robert S Molday for the generous gift of a Rho1D4-producing hybridoma and Dr. Keita Sato for valuable discussion of the manuscript and technical advice about the cAMP assay. Funding: This work was supported in part by Grants-in Aid for Scientific Research of MEXT to YS (16H02515), YI (19K21848), and TY (16K07437), CREST, JST JPMJCR1753 (TY), a grant from the Kyoto University Foundation (TY) and a grant from the Takeda Science Foundation (TY).

Senior Editor

  1. Richard W Aldrich, The University of Texas at Austin, United States

Reviewing Editor

  1. Andrew C Kruse, Harvard Medical School, United States

Reviewers

  1. Peter Hegemann, Humboldt University of Berlin, Germany
  2. Gebhard Schertler, Paul Scherrer Institute, Switzerland

Publication history

  1. Received: November 30, 2021
  2. Preprint posted: December 6, 2021 (view preprint)
  3. Accepted: February 14, 2022
  4. Version of Record published: February 24, 2022 (version 1)

Copyright

© 2022, Sakai et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Kazumi Sakai
  2. Yoshinori Shichida
  3. Yasushi Imamoto
  4. Takahiro Yamashita
(2022)
Creation of photocyclic vertebrate rhodopsin by single amino acid substitution
eLife 11:e75979.
https://doi.org/10.7554/eLife.75979

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    Age-dependent loss of body wall muscle function and impaired locomotion occur within 2 weeks in Caenorhabditis elegans (C. elegans); however, the underlying mechanism has not been fully elucidated. In humans, age-dependent loss of muscle function occurs at about 80 years of age and has been linked to dysfunction of ryanodine receptor (RyR)/intracellular calcium (Ca2+) release channels on the sarcoplasmic reticulum (SR). Mammalian skeletal muscle RyR1 channels undergo age-related remodeling due to oxidative overload, leading to loss of the stabilizing subunit calstabin1 (FKBP12) from the channel macromolecular complex. This destabilizes the closed state of the channel resulting in intracellular Ca2+ leak, reduced muscle function, and impaired exercise capacity. We now show that the C. elegans RyR homolog, UNC-68, exhibits a remarkable degree of evolutionary conservation with mammalian RyR channels and similar age-dependent dysfunction. Like RyR1 in mammals, UNC-68 encodes a protein that comprises a macromolecular complex which includes the calstabin1 homolog FKB-2 and is immunoreactive with antibodies raised against the RyR1 complex. Furthermore, as in aged mammals, UNC-68 is oxidized and depleted of FKB-2 in an age-dependent manner, resulting in ‘leaky’ channels, depleted SR Ca2+ stores, reduced body wall muscle Ca2+ transients, and age-dependent muscle weakness. FKB-2 (ok3007)-deficient worms exhibit reduced exercise capacity. Pharmacologically induced oxidization of UNC-68 and depletion of FKB-2 from the channel independently caused reduced body wall muscle Ca2+ transients. Preventing FKB-2 depletion from the UNC-68 macromolecular complex using the Rycal drug S107 improved muscle Ca2+ transients and function. Taken together, these data suggest that UNC-68 oxidation plays a role in age-dependent loss of muscle function. Remarkably, this age-dependent loss of muscle function induced by oxidative overload, which takes ~2 years in mice and ~80 years in humans, occurs in less than 2–3 weeks in C. elegans, suggesting that reduced antioxidant capacity may contribute to the differences in lifespan among species.